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. 2015 Apr 10;8:61.
doi: 10.1186/s13068-015-0231-1. eCollection 2015.

A Microbial Platform for Renewable Propane Synthesis Based on a Fermentative Butanol Pathway

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Free PMC article

A Microbial Platform for Renewable Propane Synthesis Based on a Fermentative Butanol Pathway

Navya Menon et al. Biotechnol Biofuels. .
Free PMC article

Abstract

Background: Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli.

Results: We report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L).

Conclusions: This study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde. Graphical abstractPropane synthesis derived from a fermentative butanol pathway is enabled by metabolic engineering.

Keywords: Aldehyde deformylating oxygenase; Butanol; Cyanobacteria; Escherchia coli; Microbial pathway engineering; Propane.

Figures

Graphical abstract
Graphical abstract
Propane synthesis derived from a fermentative butanol pathway is enabled by metabolic engineering.
Figure 1
Figure 1
The CoA-dependent butanol pathways used for the production of propane in E. coli. The four CoA-dependent butanol producing synthetic routes (atoB-adhE2 route, atoB-TPC7 route, nphT7-adhE2 route and nphT7-TPC7 route) explored for butanol production in E. coli are shown. AdhE2, aldehyde-alcohol dehydrogenase; ADO, aldehyde deformylating oxygenase; Ahr, aldehyde reductase; AtoB, acetyl-CoA acetyltransferase; CAR, carboxylic acid reductase; Crt, 3-hydroxybutyryl-CoA dehydratase; Hbd, 3-hydroxybutyryl-CoA dehydrogenase; NphT7, acetoacetyl CoA synthase; Ter, trans-2-enoyl-CoA reductase; YciA, acyl-CoA thioester hydrolase.
Figure 2
Figure 2
Total butanol produced by the engineered E. coli BL21 strains . Total butanol concentration obtained after 72 h of cultivation of E. coli wild-type cells harbouring engineered constructs is shown. The experimental protocol is described in the ‘Materials and methods’ section. Error bars are standard deviation (n = 4).
Figure 3
Figure 3
Total propane produced by the engineered E. coli BL21 strains . Total propane accumulated over 4 h of reaction under assay conditions performed in GC vials for the pathway-engineered E. coli cells, after overexpressing with ADO. The experimental protocol is described in the ‘Materials and methods’ section. Error bars are standard deviation (n = 4). ADO, aldehyde deformylating oxygenase; IPTG, isopropyl β-D-1-thiogalactopyranoside.
Figure 4
Figure 4
Propane production in pathway-engineered E. coli strains containing wild-type ADO or the ADO A134F variant enzyme. E. coli cells were either pathway-engineered to include the atoB-TPC7 route (indicated by red colour) or the nphT7-TPC7 route (indicated by green colour) and contain wither wild-type ADO or the ADOA134F variant enzyme in the absence/presence of ferredoxin (Fdx) from Synechocystis sp. PCC 6803. A detailed protocol for the pathway engineering and propane detection is included in the ‘Materials and methods’ section and with the supporting information (Additional file 1: Figure S6). Error bars are standard deviation (n = 4).
Figure 5
Figure 5
Propane produced in pathway-engineered Δahr/ΔyqhD single or double knockout E. coli strains and the effects of co-expressing a ferredoxin electron donating system. Propane production in the pathway-engineered ΔyqhD knockout cells with wild-type ADO or with the ADOA134F variant enzyme is shown (A). The ΔyqhD knockout strains were either engineered to contain the atoB-TPC7 route (indicated by red colour) or the nphT7-TPC7 route (indicated by green colour). Wild-type ADO or the ADOA134F variant enzyme was co-expressed in the engineered cells either in combination with or without ferredoxin (Fdx) from Synechocystis sp. PCC 6803 (B). A detailed protocol for the pathway engineering and propane detection is included in the ‘Materials and methods’ section and supporting information (Additional file 1: Figure S6). Error bars are standard deviation (n = 4). ADO, aldehyde deformylating oxygenase.
Figure 6
Figure 6
Larger scale cultures of the best performing propane-producing pathways . The atoB-TPC7 route (indicated by red colour) and nphT7-TPC7 (indicated by green colour) engineered in ΔyqhD knockout cells in the presence of the ADOA134F variant and ferredoxin system were analysed at larger scale. The culture volume was scaled up to 400-fold to 200 mL, in a 300-mL flask sealed with airtight rubber septum. The propane accumulation for 12 h is shown. Error bars are standard deviation (n = 3).
Figure 7
Figure 7
The plasmid design used to construct engineered propane producing pathways in E . coli. The structure of all plasmids used in this study for E. coli pathway engineering is shown. The preparation and design of these plasmids are described in the ‘Materials and method’ section.

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